PubMed Health⌕ Search

Biomedical subjects

J L Werth

Publications and source records attributed to J L Werth.

11 recordsLinked to original sources
PubMed1996

Modulation of calcium efflux from cultured rat dorsal root ganglion neurons.

The free intracellular Ca2+ concentration ([Ca2+]i) is governed by the balance between the activation of Ca2+ channels and buffering and efflux processes. We tested the hypothesis that Ca2+ efflux pathways are susceptible to modulation. The whole-cell patch-clamp technique was used in combination with Indo-1-based microfluorometry to record Ca2+ current and [Ca2+]i simultaneously from single rat dorsal root ganglion (DRG) neurons grown in culture. Depolarizing test pulses (-80 to 0 mV, 100-300 msec) elicited [Ca2+]i transients that recovered to basal levels by a process best-fit with a single exponential (tau = 5.1 +/- 0.4 sec; n = 14) and were independent of Ca2+ load (40-500 pC) over this range of test pulses. [Ca2+]i transients recorded in whole-cell configuration were similar to those elicited by a brief train of action potentials in unclamped neurons. Inhibition of Ca2+ sequestration into intracellular stores with thapsigargin had no effect on the kinetics of recovery. Inhibition of plasma membrane Ca2+ ATPase (PMCA) function by including a peptide inhibitor (C28R2) in the patch pipette significantly slowed recovery to basal [Ca2+]i (tau = 9.9 +/- 0.8 sec; n = 4). Preincubation with calmidazolium, a calmodulin antagonist, produced modest slowing of Ca2+ efflux. Phorbol dibutyrate, an activator of protein kinase C (PKC), accelerated Ca2+ efflux only when the PMCA had been inhibited by C28R2. We conclude that in DRG neurons PMCAs are responsible for lowering [Ca2+]i after small Ca2+ loads and that PMCA-mediated Ca2+ efflux is modulated by calmodulin- and PKC-signaling pathways.

Action Potentials↗
PubMed1995

Empirically based criteria for rational suicide: a survey of psychotherapists.

This qualitative study was designed to develop a set of empirically based criteria for rational suicide by asking psychotherapists to define "rational suicide." Data analysis revealed three components of a rational suicide: (1) the presence of an unremittingly hopeless condition (eight examples provided), (2) a suicidal decision made as a free choice, and (3) the presence of an informed decision-making process (five elements listed). It is suggested that the criteria may possibly be used in assessing the rationality of suicidal clients.

Attitude of Health Personnel↗
PubMed1994

2',3'-Dideoxycytidine alters calcium buffering in cultured dorsal root ganglion neurons.

Mitochondria play a prominent role in shaping intracellular calcium concentration ([Ca2+]i) transients in dorsal root ganglion neurons. Mitochondrial DNA polymerase is inhibited by antiviral compounds such as 2',3'-dideoxycytidine (ddC). Here, we test the hypothesis that ddC can alter mitochondrially mediated Ca2+ buffering in neurons. Chronic treatment of dorsal root ganglion cultures with ddC (1 microM) lowered mitochondrial DNA levels and decreased the mitochondrially mediated component of depolarization-induced [Ca2+]i transients. The inhibition increased in a time-dependent manner, reaching a maximum at 6 days. ddC did not affect small, action potential-evoked, [Ca2+]i transients that are predominantly buffered by Ca(2+)-ATPases, suggesting that ATP levels were not depleted. The drug did not inhibit whole-cell Ca2+ currents, indicating that the Ca2+ load was not affected. Thus, ddC produces a graded, time-dependent inhibition of mitochondrial function that is reflected, in part, by a decrease in the direct buffering of Ca2+ by mitochondria. This effect may contribute to the peripheral neuropathy that results from ddC treatment. Furthermore, ddC promises to be a useful tool to study the role of mitochondria in [Ca2+]i homeostasis and neurodegenerative processes.

Animals↗
PubMed1994

Mitochondria buffer physiological calcium loads in cultured rat dorsal root ganglion neurons.

We sought to determine whether low-affinity, high-capacity mitochondrial Ca2+ uptake contributes to buffering physiological Ca2+ loads in sensory neurons. Intracellular free calcium concentration ([Ca2+]i) and intracellular free hydrogen ion concentration ([H+]i) were measured in single rat dorsal root ganglion (DRG) neurons grown in primary culture using indo-1 and carboxy-SNARF-based dual emission microfluorimetry. Field potential stimulation evoked action potential-mediated increases in [Ca2+]. Brief trains of action potentials elicited [Ca2+]i transients that recovered to basal levels by a single exponential process. Trains of > 25 action potentials elicited larger increases in [Ca2+]i, recovery from which consisted of three distinct phases. During a rapid initial phase [Ca2+]i decreased to a plateau level (450-550 nM). The plateau was followed by a slow return to basal [Ca2+]i [Ca2+]i transients elicited by 40-50 action potentials in the presence of the mitochondrial uncoupler carbonyl cyanide chlorophenyl hydrazone (CCCP), or the electron transport inhibitor antimycin A1, lacked the plateau, and the recovery to basal [Ca2+]i consisted of a single slow phase. Depolarization with 50 mM K+ produced a multiphasic [Ca2+]i transient and increased [H+]i from 74 +/- 3 to 107 +/- 8 nM. The rise in [H+]i was dependent upon extracellular Ca2+ and was inhibited by mitochondrial poisons. With mitochondrial Ca2+ buffering pharmacologically blocked, the recovery to basal [Ca2+]i was unaffected by removal of extracellular Na+. We conclude that large Ca2+ loads are initially buffered by fast mitochondrial sequestration that effectively uncouples electron transport from ATP synthesis, leading to an increase in [H+]i. Small Ca2+ loads are buffered by a nonmitochondrial, Na(+)-independent process.

Action Potentials↗
PubMed1992

Lipophilic amino alcohols with calcium channel blocking activity.

A series of novel lipophilic amino alcohols, analogs of the anticholinergic drug vesamicol, were evaluated for Ca2+ channel blocking activity. The effects of these drugs on depolarization-induced intracellular free Ca2+ concentration ([Ca2+]i) transients were examined in single NG108-15 cells and dorsal root ganglion (DRG) neurons in culture. [Ca2+] was recorded with the Ca2+ indicator Indo-1 and a dual emission microfluorimeter. Structure-activity studies indicated that features required for Ca2+ channel blocking activity were distinct from those required for anticholinergic activity. In particular, the Ca2+ channel blocking activity was insensitive to the configuration at the chiral center, whereas the anticholinergic activity was clearly enantioselective. One of the most active compounds, 3-(3-bromophenyl)-2-hydroxy-1-[1-(4-phenylpiperidinyl)]propane (2b), was characterized in more detail. This compound inhibited the dihydropyridine-sensitive Ca2+ channel response in NG108-15 cells, evoked by depolarization with 50 mM K+, with an IC50 of 5 microM. Field potential stimulation of DRG neurons elicited [Ca2+]i transients mediated by at least three Ca2+ channel subtypes; compound 2b inhibited the entire Ca2+ channel response with an IC50 of 1 microM. A key element required for Ca2+ channel blocking activity was the presence of an electron withdrawing substituent on the pendant phenyl ring. Modification of the amino alcohol structure may lead to more potent compounds with broad spectrum Ca2+ channel blocking activity. These structures provide a new chemical starting point for the development of Ca2+ antagonists.

Amino Alcohols↗
PubMed1991

omega-Conotoxin exerts functionally distinct low and high affinity effects in the neuronal cell line NG108-15.

The Ca2+ channel blockade produced by omega-conotoxin GVIA (omega-CgTx) was studied in single, forskolin-differentiated, NG108-15 cells, using dual-emission microfluorimetry and the whole-cell patch-clamp technique. Whole-cell currents through Ca2+ channels were measured with 5 mM Ba2+ as the charge carrier. Application of 1 microM nitrendipine inhibited by 90% the currents evoked by stepping from -30 mV to 0 mV. Omega-CgTx (1 microM) inhibited these currents by 28%. These data suggest the possibility that NG108-15 cells express two types of dihydropyridine-sensitive Ca2+ channel, one sensitive and the other insensitive to blockade by omega-CgTx. The nature of the Ca2+ channel blockade produced by these agents was studied further, using depolarization-induced intracellular free Ca2+ concentration [( Ca2+]i) transients recorded with the Ca2+ indicator indo-1 and a dual-emission microfluorimeter. A 30-sec superfusion with 50 mM K+ increased the [Ca2+]i from a basal level of 142 +/- 10 nM to a peak level of 1655 +/- 287 nM. This [Ca2+]i transient was blocked completely and reversibly by nitrendipine, in a concentration-dependent manner (IC50 = 1.9 nM). In contrast, omega-CgTx produced a maximal inhibition of the depolarization-induced rise in [Ca2+]i of only 52% in the presence of physiological concentrations of divalent metals. The block was irreversible. This inhibition was concentration dependent until the point of maximal inhibition, at which point the channel block reversed in a graded manner. This entire U-shaped dose-response curve could be shifted in a parallel fashion by modulation of the extracellular divalent metal concentration, without changes in the maximal inhibition. Repeated applications of or prolonged incubations with omega-CgTx failed to increase the maximal block. Treatment with a high (1 microM) concentration of omega-CgTx, which produced a modest (10%) inhibition of Ca2+ influx, protected the cell from a second exposure to a normally effective concentration of omega-CgTx (10 nM). Depolarization-induced [Ca2+]i transients in cells treated with 10 nM toxin were inhibited by 45%, and this inhibition could not be reversed by subsequent exposure to a high concentration of omega-CgTx. We conclude that there are two omega-CgTx binding sites on these cells, one to which omega-CgTx binds with high affinity, producing an irreversible Ca2+ channel blockade, and a second to which omega-CgTx binds with lower affinity. Binding to this second site is irreversible and does not block the channel but does prevent access to the high affinity site.(ABSTRACT TRUNCATED AT 400 WORDS)

Calcium↗